QUALITY TESTS FOR PROTEIN MEALS
Dr. Jowaman Khajarern and Dr. Sarote Khajarern
Department of Animal Science, Faculty of Agriculture
Khon Kaen University, Thailand
the early 1970s, the feed industry in Southeast Asia has made
tremendous expansion. High quality materials especially protein
meals (fish meal and soybean meal) are often in short supply
and also exhibit a variation in the quality aspects of nutrient
availability. Since protein sources especially fish meal and
soybean meal generally have high unit cost, the company must
establish written ingredient quality standards for purchasing,
but the methods for examining the physical qualities, especially
for foreign materials and evidence of mold, must be fast,
accurate and practical by the operators at the receiving plant.
The operators must be trained to recognize and understand
the quality of raw material to perform their visual and other
quick physical and chemical examinations, and in the proper
methods of sampling. The objective of this paper is to highlight
the most important method of quick tests for protein meals
used for the purchasing of the raw materials.
Sources of Protein Meals for Non-ruminants and Aquaculture
Sources Plant Protein Animal Protein
Conventional Major Soybean meal Fish meal
Fullfat soybean Meat meal
Sunflower meal Meat and bone meal
Sesame meal Milk products
Peanut meal Poultry byproducts
Canola and Rapeseed meal
Corn gluten meal
Palm kernel meal
Minor Soya protein isolate Feather meal
Wheat gluten meal Blood meal
Mung bean Marine soluble products
Potato protein Shrimp products
Cotton seed meal Squid products
Kapok seed meal Yeast
Non-conventional Rubber seed meal Leather meal
Lupins Silk worm pupa meal
Safflower meal Crab products
Linseed meal Lard pulp
Field peas Brewer's byproducts
Mustard meal Distiller's byproducts
Cocoa seed meal
Quality Control in Different Protein Meals
Ingredient Quality Physical characteristics (analyst's skills): color, texture, odor and
(Qualitative) taste, particle size (screen analysis), shape, adulteration, damage
and deterioration, bulk density, spot and quick chemical tests.
Ingredient Quality Proximate analysis: moisture, CP, CF, EE, NFE, ash, silica or
(Quantitative) sand, salts etc.
Protein quality: protein solubility or dispersibility, bitrogen
solubility, mailard reaction product, biogenic amines, trypsin
inhibitor activity, urease activity, dye binding, pepsin
digestibility, urea and nonprotein nitrogen.
Amino acid: composition, digestibility, availability
- Extrinsic (contaminants): mycotoxins, insects, weeds,
insecticide, herbicides, fungicides.
- Intrinsic: allergins, lectins, phytoestrogens,
glucosinolate, saponins, tannins, ricin, sinapine,
Decomposition and rancidity test: acid value, peroxide value, etc.
Test for Fish Meal Quality Control
fish meal is produced from two types of raw materials, fish
wastes from human food industry and whole fish. Differences
in composition of fish meal may be attributed to many causes:
variation in raw materials, differences in processes, contamination
of raw materials with some waste products or with sand, impurities,
excessive salts and/or fat content or excessive moisture,
adulteration with other sources of meal, meat and bone meal,
plant proteins, etc. Fish meal is also prone to contamination
with biogenic amine and gizzarosine found during processing
and storage that have been allowed to spoil or putrefy and
have dramatic impact on the quality and nutritive composition
of fish meal. The main aspects of quality control for consideration
material type: whole fish or trimmings
temperature: low temperature (LT) high temperature (HT)
on digestibility of protein and over-heated by using racemization
of aspartic acid (Luzanna et al., 1996) to indicate digestibility
of protein and a simple dilute pepsin test (strength 0.0002
%) at 450C will identify overheated fish meal.
of raw material: Fish spoils protein breakdown to amines
(histamine, cadaverine putrescine and tyramine). The sum
of the four should be less than 2,000 ppm or predominant
in anchovy, mackerel, sardine, etc. or cadaverine predominant
in capelin, sandeel, sprat, etc).
quality: Oxidation of fat to free fatty acids given
a crude measure of fish freshness. Oxidation of fat can
reduce growth of animal and aquaculture. Ethoxyquin has
been found to be the most effective antioxidant. Free fatty
acid (FFA) peroxide value (PV) and quick test for rancidity
should be assessed for quality control.
standards: Fish meal should be free of salmonella and
mycotoxin. Because carbohydrate or starch is absent, mold
growth generally does not occur if fish meal is properly
stored in dry conditions and stabilized with antioxidants.
Test for Quality Grade of Fish Meal
10g of fish meal in a 100 ml tall form beaker and pour 80
ml carbon tetrachloride into the beaker, stir and allow
to settle (5 min.).
off the floating organic fraction with stainless steel spoon
into filter paper. Clean off the side of the beaker and
spoon and then pour off the liquid to separate floating
and the submerged fraction into the filter paper.
the two fractions into a 1100C oven for 10 min.
then allow to cool and weigh each fraction.
approximate percentage of organic (fish flesh) and inorganic
(fish bone) may be calculated.
Shrimp and Salmon Grade Inorganic Fraction
Fish meal Grade A (CP>70%) <12%
Fish meal Grade B (CP>65%) <16%
Fish meal Grade C (CP>62%) <20%
Animal Grade Inorganic Fraction
Fish meal Grade A (CP>58%) <30%
Fish meal Grade B (CP>35-58%) <35%
Fish meal Grade C (CP>50-55%) <40%
Fish and bone meal (CP 35-50%) >40%
Test for Quality Grade of Fish Meal
adulterants such as (ammonium sulfate, ammonium phosphate,
ammonium nitrate, etc) are non-protein nitrogen and are soluble
in water. When they react with mercuric potassium iodide alkaline
solution mixture, a heavy orange precipitate color occurs.
mercuric chloride is made into a thin paste with a little
water, 3.5 g potassium iodide is added and solution takes
place. 12 g sodium hydroxide is dissolved in 50 ml. Water
is added and the whole made up to 100 ml. The rather turbid
solution is allowed to stand for several days, decanted and
stored in a brown bottle.
2-3 g of test sample in a 100 ml beaker and add 10-15 ml
distilled water, then stir and let stand for 2-3 min.
3-5 drops of test sample into white porcelain spot plates
and add 2-3 drops of mercuric-potassium iodide alkaline
non-protein nitrogen is present, a heavy orange precipitation
color appears. The intensity of orange precipitation depends
on the amount of non-protein nitrogen present.
Test of Hydrolyzed Feather Meal from Fish Meal
feather meal contains a high percentage of cystine (6-7%).
When the sample is digested with sodium hydroxide, the cystine
and cysteine are liberated and reaction with lead acetate
gives the dark brown black color on the surface of the particle.
A: Sodium hydroxide 10%
B: Dissolve 50 g of lead acetate in 800 ml water, then add
20 ml glacial acetic acid, stir and pour 20 ml glucerol, shake
and dilute with water to 1,000 ml.
one teaspoon of well mixed standard hydrolyzed feather meal
and test sample of fish meal into two sets of petri dishes.
10-15 ml of solution A into all the two sets of each test
sample. Swirl gently to spread samples evenly in each dish
and let them stand for 10 min.
10-15 ml of solution B into each first set of petri dishes
and into the second set add 10-15 ml distilled water. Mix
gently by turning around each petri dish and let them stand
again for 10 min.
standing, a visible browning reaction color develops until
black colored particles appear in the first set of petri
dish for the standard hydrolyzed feather meal. When compared
to the second set (without adding solution B), no visible
brown color develops after 10 mins.
the test fish meal sample with the standard hydrolyzed feather
meal sample and also the visible browning color between
the first and second set of each test sample. If the color
of these two sets differs, the fish meal is adulterated
with hydrolyzed feather meal.
Test of Hydrolyzed Leather Meal from Fish Meal
meal is waste by-products from hide trimmings which contain
chromium salts. When a sample is ashed, the visible dark green
chromium salts develops. Diphenyl carbazide is an excellent
reagent to react with chromium in dilute sulfuric acid (0.1
MH2SO4) which produces a violet color
and will fade within 10-15 mins.
A: (0.1 MH2SO4
Solution B: Dissolve 1-2 g diphenyl carbazide in 100 ml distilled
fish meal test sample at 6000C for 2 hrs and
examine for the presence of dark green chromium salts. Dark
green particle of ash is a conducive evidence of the adulteration
of leather meal in the sample.
confirm the presence of chromium, place a small amount of
ash into a white spot plate. Add 2-3 drops of solution A
and then add 2-3 drops of solution B.
- A red-violet
color indicates the presence of chromium.
Test for Animal and Marine Products
animal and marine products spoil, protein breaks down to amines.
The residue of these biogenic amine can indicate the freshness
or decomposition of the sample. If the sample is badly decomposed,
the test sample will darken quickly with saturated lead acetate
paper and it is not suitable for feeding.
sulfuric acid: 5 ml of conc. H2SO4 to
45 ml H2O
lead acetate solution.
5 g of test sample into 250 ml erlenmeyer flask.
a cork, which fits tightly, with a 2" x ¼"
strip of white filter paper pinned to the bottom, moistened
with saturated lead acetate.
50 ml dilute sulfuric acid into the sample then immediately
insert the cork and let it stand in a warm room for 16 hrs.
the sample is badly decomposed, the test paper will darken
Test for Identification of Plant Protein and Animal Protein
from plants contain starch and cellulose. When it reacts with
iodine and chlor-zinc iodine solution, the starchy tissue
releases a blue color and the plant fiber or cellulose develops
a purple-brown color when examined under a microscope.
solution: 0.5 g I and 1.5 g KI dissolved in 25 ml distilled
solution: Dissolve 100 g ZnCl2 in
60 ml water, then add 20 g KI and 0.5 g I.
1-2 g test sample with 100 ml boiling water or boil the
mixture for 2-3 min. Place a few ml of the cooled mixture
on a spot plate or test tube and add 5-6 drops of iodine
solution. If starch is present, the mixture turns blue.
1-2 g test sample into a petri dish. Add 5-6 drops of chlor-zinc
iodine solution and let stand for 10 min. A purple brown
color indicates the presence of plant fiber, whereas yellow
indicates animal fiber (protein) using a microscopic examination.
Considerations for Soybean Meal
meal is a major plant protein source for poutlry, livestock
and aquaculture. Soybean meal used in Asian countries exists
in several forms, with solvent extracted material containing
hulls being the most common. Many locations use imported soybean
meal from USA, Brazil, Argentina and India. Full fat soybean
is also available, produced by extrusion or dry roasting in
small scale plants. The following are specifications for soybean
meal and dehulled soybean meal (Hi-Pro).
Density Range: 57-64 g/100 cc for both soybean meal and
Analysis: 95-100% through US #10 screen; 40-60% through
US #20, 6% maximum through US #80 for both soybean and soybean
Physical Properties for both soybean and soybean (Hi-Pro).
Light tan to light brown
Fresh, not musty, not sour or burned
Homogeneous, free flowing, no lumps or cakes, without
coarse particles or dusty
Bland and free of any beany or burned taste
actvity: 0.05-0.20 pH unit change for both soybean meal
and soybean (Hi-Pro).
(maximum): 12% for both soybean meal and soybean (Hi-Pro).
solubility 0.2% KOH: 73-85% for both soybean meal and soybean
Dispersibility Index: 15-30% for both soybean meal and soybean
Solubility Index: 15-30% for both soybean meal and soybean
Particularly check for urea, non-protein nitrogen or ammonia
for both soybean meal and soybean (Hi-Pro).
(minimum): 44.0% for soybean meal; 47.5-49% for soybean
(minimum): 0.5% for both soybean meal and soybean (Hi-Pro).
(maximum): 7.0% for soybean meal; 3.3-3.5% for soybean (Hi-Pro).
(maximum): 6% for both soybean meal and soybean (Hi-Pro).
(maximum): 2% for both soybean meal and soybean (Hi-Pro).
((kcal/kg): Approximately 2,375 for soybean meal; 2,525
for soybean (Hi-Pro).
test for Animal Products and Oilseed Meal
chloroform mixture (6:4): Glacial acetic acid 60 ml and
40 ml chloroform.
5g test sample into 250 ml Erlenmeyer flask.
50 ml of acetic chloroform mixture and 1 ml saturated potassium
iodide and shake.
50 ml distilled water and starch indicator, then shake again.
of blue color indicates rancidity.
Phloroglucinal solution in ether.
10 ml of oil or melted fat sample with 10 ml of 0.1% phloroglucinal
solution and 10 ml of conc. Hydrochloric acid for 20 sec.
A pink color indicates incipient rancidity.
oil is diluted 1 in 20 kerosene and the test is still positive,
the rancidity will probably be evident to the taste and
Test for Urea (Qualitative Test)
solution: 0.2 g urease stir into 10 ml H2O.
blue solution: Rub 0.15 g Bromothymol blue in mortar with
2.4 ml 0.1 N NaOH, wash mortar and pestle with H2O
and dil. To 50 ml H2O.
paper A: Mix 10 ml urease solution 1 and 10 ml indicator
soln. 2. Pour mixture into watch glass, dip pieces of filter
paper (Whatman No. 5) in soln. And hang paper to dry. Store
dry paper (Orange color) in well-stoppered dark glass bottle
in a cool place.
paper B: Dilute indicator soln. 2 with equal portion with
H2O. Dip pieces of filter paper (same kind used
for test paper A) in indicator soln. And hang to dry as
2-3 g of test sample in 50 ml H2O and let it
stand for 2-3 min.
2-3 drops of test sample on dry test paper A, the appearance
of blue or green spot after a few minutes of incubation
at room temperature indicates urea.
detection of urea in a very small, dry particle, dip both
test paper A and B H2O and then shake the papers
to remove excess H2O by using clean tweezers.
Place the papers on a clean flat piece of glass. Place the
sample on the papers and cover with another clean flat piece
of glass by pressing down gently. Blue spots on the test
paper A indicate urea (30-60 sec.). Spots continue to develop
and enlarge for 10-20 min. and then fade gradually. Time
varies inversely with urea concentration. If blue spots
develop on both paper A and B, this indicates alkaline particles.
Identification of Protein Meals
microscopy experts must be fast and accurate to obtain quality
assurance. Adulterants and contaminants in both ingredients
and finished feeds are best detected microscopically. Special
training on description and characteristics of feed ingredients
are the keys for rapid microscopic identification.
the feed is mashed, use about a teaspoonful of feed for sieving.
Rub the feed in a mortar with a pestle if the feed is pelleted
or crumbled. When the sample is reduced to a mash-like consistency,
it is divided in three sets of sieves (20, 40, 60 mesh), and
thus end up with four fractions of the feeds. Each fraction
is put in a glass petri dish cover. The sample is now ready
for microscopic examination. Using a stereomicroscope, scan
each particle in the first and second dishes. The feed in
the third and fourth dishes is used for high power compound
microscope and also for quick chemical spot test. For a clearer
observation of plant histology and microscopic appearance,
the feed is heated with 8% KOH steam bath for 30-45 min. If
this treatment is not satisfactory, treat the fresh portion
for a short time by gently heating with acidified chloral
hydrate glycerol solution.
characteristics on Microscopic Identification of Protein Meals
(Contact us for a copy
of the annex picture).
Products: Orange pigmentation, segmented antennae with
calcareous shell and will effervesce in diluted HCI, honey
combed round cells on the outer layer of shell.
Products: Curved scales with concentric rays, bone sexhibit
lacunae with well defined canaliculi, milky glass beads
with broken surface eye lenses.
Meal: Segmented leg and antennae, thin shell with mica-like
and in some areas, may appear cross-hatch type of marking,
feathery delicate gill tissue, amber colored cells of compound
Products: Mottled body fragment with black pigment spots,
tentacles or sucker pieces present, no lacunae or surface
lines on internal shell fragments.
Meal: Spherical particle, smooth surface with glass
when rubbed, dark red to almost black in color.
Meal and Meat and Bone Meal: Strong greasy odor, consist
of hoof, horn, hair, fluff and vegetable fiber, cylindrical
rods smooth muscle with alternative dark and light striated
Meal: Yellow to brown oval hilium with a clear slit,
pox-marked outer surface hull, hourglass and palisade cells
from the hull are the major cellular keys for soybeans and
also elongated cells below the peripheral cells of the cotyledon.
Meal: Thin skin with copper to red color, higly pitted
cell of pod fragment and the lack of palisade cells in the
testa, elongated pite in hypodermal stone cells and unique
cross fiber cell of pod.
Meal: Striped or all black varieties for the hull, leathery
hull with a paper-like lining. Twin hairs, united almost
to their tips on the outer surface of the cypsela, unbroken
pericarp fragment may appear as broken pieces in the medium,
the outer epiderm of transversely elongated cells with zigziag
Meal: Many species of rape are lumped together and are
difficult to identify separately by structured features.
For all practical purposes, the examination of the seed
coat or testa for degree of reticulation is important. The
inner surface seed coat has a delicate semi-transparent,
white sheet adhering to the surface.
Meal: Seed coat or outer epidermal cells contain calcium
oxalate crystals with black brown or yellow brown colored
and granular surface.
Meal: Long, flat and twisted fibers adhering to the
hulls, kernel fragments are yellow to brown containing many
round, red, brown gossypol glands. The hull edge has a light
brown layer with stairstep facets. The epidermal cells are
heavy walled with dark pigmented interiors. Palisade cells
can also be used for identification.
Meal: Irregularly shaped flaky fragment with large,
colorless, straight, thin walled cells of endosperm containing
of Official Analytical Chemists, 1984. Official Methods
of Analysis. 12th Edition, AOAC, Box 540, Benjamin
Franklin Station, Washington DC 20044.
J., D. Sinchermsiri, and A. Hanbunchong, 1987. Manual Feed
Microcopy and Quality Control. Dhornhvaj Co., Ltd. Bangkok,
U., T. Mentasti, V. Moretli, A. Albertini, and E. Valfre,
1996. Aspartic acid raccimization in fish meal as induced
by thermal treatment. Aquac. Nutr. 2: 95-99.
of Microscopic Analysis of Feeding Stuffs, 1966. The American
Association of Feed Microscopists.
of of Microscopial Analysis of Feedstuff, 1992. 3rd
Edition, The American Association of Feed Microscopists.
D., 1970. The Chemical Analysis of Foods, 6th
Edition. J & A Churchill, 164 Gloucester Place, London.
541 Orchard Road #11-03 Liat Towers
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MITA (P) NO. 096/11/97 (Vol. FT45-1998)